The invention relates to the administration of urokinase-type plasminogen activators ("u-PA"), such as pro-urokinase ("pro-UK"), to patients.
Thrombolytic therapy, e.g., with streptokinase ("SK") or tissue-type plasminogen activator ("tPA"), is widely used to dissolve potentially life-threatening blood clots, e.g., after acute myocardial infarction. Angioplasty, e.g., percutaneous transluminal coronary angioplasty ("PTCA"), is used to open coronary artery stenoses. Such therapies are associated with an undesirably high rate of reocclusion, and often reinfarction, which can occur within hours after successful lysis, and substantially attenuates the therapeutic effect. For example, reocclusions occur in about 29% of patients treated with tPA, Morrie et al., Am Heart, 122: 375-380 (1991), Kalbfleisch, et al., Am. J. Cardiol., 69: 1120-1127 (1992), and in about 10% of patients treated with SK, Yusuf et al., J.A.M.A., 260: 2088-2093 (1988). In addition, about 30% of the stenoses opened by PTCA reocclude within three months.
To counteract such undesirable reocclusions, various adjunctive therapies have been developed for use after thrombolysis. For example, aspirin, heparin, thrombin inhibitors, platelet inhibitors, monoclonal antibodies to platelet glycoprotein IIb/IIIa, and activated protein C have all been identified as potential agents for use in adjunctive strategies. In addition, .beta.-blockers, calcium antagonists, angiotensin-converting enzyme inhibitors, and nitrates also may be useful adjuncts to thrombolytic therapy.
Adjunctive agents such as high dose heparin, hirudin analogs, or anti-platelet agents can be associated with hemorrhagic complications, e.g., systemic bleeding. Moreover, these adjunctive therapies are expensive and complicate thrombolytic therapy, thereby compromising optimal early administration of the thrombolytic agents.
One such thrombolytic agent is pro-UK, or urinary-type plasminogen activator, which is a naturally occurring plasminogen activator consisting of a single-chain polypeptide made up of 411 amino acids; hence, pro-UK is also referred to as single chain urokinase-plasminogen activator ("scu-PA"). Pro-UK was first purified from urine, see U.S. Pat. No. Reissue 32,271, and has subsequently been produced by recombinant techniques. Pro-UK is a pro-enzyme which is converted into urokinase ("UK") by the action of plasmin. Once it enters the bloodstream, pro-UK is rapidly cleared from the plasma, and as described in, e.g., Collen et al. Am. J. Cardiol., 60: 431-434 (1987), has a half-life (T.sub. 1/2) of about eight minutes. Therefore, when used for therapeutic purposes, pro-UK is typically administered by infusion which, when terminated, is followed by rapid clearance of the drug from the plasma.
Pro-UK has been used in clinical trials for therapeutic thrombolysis, e.g., in patients with myocardial infarction, pulmonary embolism, or deep vein thrombosis. For example, U.S. Pat. No. 5,055,295 (Welzel et al.) describes the lysis of blood clots with combinations of pro-UK and UK in post-operative patients, and in patients who had a myocardial infarction or deep vein thrombi.
Bolus injections of pro-UK are associated with a short plasma half-life and the absence of fibrin clot binding. However, when preceded by a bolus of UK (2.5 mg) or tPA (5 mg), the thrombolytic effect of pro-UK is synergistically promoted, and lower rates of infusion of pro-UK are efficacious (40 mg/h instead of 60-80 mg/h). See, e.g., Gurewich et al., Thromb. Res., 44: 217-228 (1986).
Bolus injections of pro-UK (0.2-2 mg/kg) have been shown in one study to produce effective thrombolysis of arterial thrombi in dogs. Badylak et al., Thromb. Res., 52: 295-312 (1988). This study showed that the thrombolytic effect in the dogs lasted for at least 30 to 40 minutes despite a plasma T.sub. 1/2 of pro-UK of only 6 minutes. The reason for this effect was not known.
In the absence of a fibrin clot, pro-UK has been reported to be relatively inert below certain concentrations in the blood. For example, Gurewich, V., Sem. Thromb. Hemostasis, 15: 123-128 (1989) has reported that "little to no fibrinogen degradation [occurred] when less than 50 mg/hour of pure wild type pro-UK was infused" in clinical trials (p. 125). In the presence of a fibrin clot, pro-UK selectively activates any plasminogen associated with the clot. However, at higher doses, non-specific plasminogen activation can occur, which converts pro-UK to UK systemically and increases the risk of bleeding.
Although exogenous pro-UK induces fibrin-specific clot lysis in plasma in vitro and in vivo, the biological role of endogenous pro-UK is believed to be related to tissue remodeling, inflammation, and cell migration rather than fibrinolysis. See, e.g., Rohrlich et al., Annual Rep. Med. Chem., 14: 229-239 (1979), and Saksela, Biochim. Biophys. Acta., 823: 35-65 (1985). Moreover, antibodies that inhibit u-PA activity fail to inhibit the spontaneous lysis of fibrin clots in plasma, which was found to be due exclusively to tPA. See, e.g., Wun et al., J. Biol. Chem., 260: 5061-5066 (1985), and Gurewich et al., Fibrinolysis, 2: 143-149 (1988). Furthermore, according to Declerck et al., Thromb. Haemostas., 67: 95-100 (1992), the normal plasma concentration of pro-UK is less than 2 ng/ml.
Park et al., Blood, 73: 1421-1425 (1989), notes that trace amounts of pro-UK are associated with the outer leaflet of sonicated platelet membranes, however, the source of this pro-UK is not identified and no studies of the incorporation of exogenous pro-UK are presented. Vaughan et al., Fibrinolysis, 4: 141-146 (1990), identifies the presence of a u-PA receptor on platelets which does not bind tPA or low molecular weight UK. This receptor has a low affinity (.about.130 nM) for u-PA and is therefore considered to be of uncertain physiological relevance, because the normal concentration of u-PA in the blood is less than about 36 pM, or about 2 ng/ml of blood.